DE69018920T2 - USE OF DNA FRAGMENTS ENCODING A SIGNAL PEPTIDE FOR SECRETION OF MATURE PROTEINS IN RECOMBINANT YEARS, EXPRESSION CASSETTES, TRANSFORMED YEARS AND METHOD FOR PRODUCING THESE PROTEINS. - Google Patents
USE OF DNA FRAGMENTS ENCODING A SIGNAL PEPTIDE FOR SECRETION OF MATURE PROTEINS IN RECOMBINANT YEARS, EXPRESSION CASSETTES, TRANSFORMED YEARS AND METHOD FOR PRODUCING THESE PROTEINS.Info
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- DE69018920T2 DE69018920T2 DE69018920T DE69018920T DE69018920T2 DE 69018920 T2 DE69018920 T2 DE 69018920T2 DE 69018920 T DE69018920 T DE 69018920T DE 69018920 T DE69018920 T DE 69018920T DE 69018920 T2 DE69018920 T2 DE 69018920T2
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Abstract
Description
Die vorliegende Erfindung betrifft neue DNA-Fragmente und ihre Verwendung als DNA-Fragmente, die für ein Signal-Peptid codieren, das geeignet ist für die Sekretion von heterologen Proteinen durch (tierische oder pflanzliche) Eukaryonten-Zellen oder Prokaryonten-Zellen (Bakterien), insbesondere Hefen, z.B. Stämme von Saccharomyces.The present invention relates to novel DNA fragments and their use as DNA fragments encoding a signal peptide suitable for the secretion of heterologous proteins by (animal or plant) eukaryotic cells or prokaryotic cells (bacteria), in particular yeasts, e.g. strains of Saccharomyces.
Bei der Herstellung eines heterologen Proteins unter Anwendung rekombinanter DNA-Verfahren ist eines der am häuf igsten verfolgten Ziele die Gewinnung (Herstellung) eines Produkts, das in das Kulturmedium der Zellen sekretiert wird, welche das heterologe Protein synthetisieren. Insbesondere im Fall der Herstellung eines Proteins von industriellem Interesse, das in großer Menge hergestellt werden soll, ist es nämlich erwünscht, daß das Protein in reifer Form, d.h. frei von jeder Aminosäure oder zusätzlichen Peptid-Sequenz, die an das Protein fusioniert geblieben ist, gebildet wird, damit es die gewünschten Eigenschaften beibehält. Darüber hinaus kann es vorteilhaft sein, daß es in das Kulturmediuin sekretiert wird, um die Gewinnungs- und Reinigungsoperationen zu erleichtern.When producing a heterologous protein using recombinant DNA techniques, one of the most frequently pursued objectives is the recovery (production) of a product which is secreted into the culture medium of the cells which synthesize the heterologous protein. In particular, in the case of producing a protein of industrial interest which is to be produced in large quantities, it is desirable that the protein be produced in mature form, i.e. free of any amino acid or additional peptide sequence which has remained fused to the protein, in order to retain the desired properties. In addition, it may be advantageous for it to be secreted into the culture medium in order to facilitate the recovery and purification operations.
Die Proteine, die durch eine Zelle, insbesondere eine Eukaryontenzelle, sekretiert werden, werden ganz allgemein in Form eines Polypeptid-Vorläufers synthetisiert, der ein Fragment, das dem reifen Protein (in der aktiven Form) entspricht, und ein als "pre"-Fragment bezeichnetes N-terminales Fragment, auch als Signal-Peptid bezeichnet, enthält, die in den Mechanismus der Sekretion des Proteins durch die Zelle eingreifen. Außerdem kann dieser Polypeptid-Vorläufer ein oder mehrere zusätzliche Fragmente, als "pro" -Fragmente bezeichnet, enthalten. In dem zuletzt genannten Fall wird der Polypeptid-Vorläufer als "pre-pro"- Vorläufer oder erster Vorläufer bezeichnet. Ein "pro"- Fragment wird in der Mehrzahl der Fälle zwischen das Signal-Peptid und das dein reifen Protein entsprechende Fragment inseriert, obgleich dies keine absolute Regel ist.The proteins secreted by a cell, especially a eukaryotic cell, are generally synthesized in the form of a polypeptide precursor, which includes a fragment corresponding to the mature protein (in the active form) and an N-terminal fragment called the "pre"fragment. Fragment, also called signal peptide, which interferes with the mechanism of secretion of the protein by the cell. In addition, this polypeptide precursor may contain one or more additional fragments, called "pro" fragments. In the latter case, the polypeptide precursor is called a "pre-pro" precursor or first precursor. A "pro" fragment is inserted in the majority of cases between the signal peptide and the fragment corresponding to the mature protein, although this is not an absolute rule.
Ein Signal-Peptid initiiert (i) die Insertion des Proteins in die Zellmembran, (ii) die Translokation des Proteins durch die Zellmembran hindurch oder (iii) den Eintritt des Proteins in das endoplasmatische Retikulum der Zelle zur Sekretion des Proteins durch das endoplasmatische Retikulum. Wenn einmal das Signal-Peptid seine Aufgabe erfüllt hat, wird es normalerweise durch proteolytische Spaltung entfernt, um ein reifes Protein oder einen zweiten Vorläufer, als "pro"-Vorläufer bezeichnet, freizusetzen, der genau wie der erste Vorläufer keine biologische Aktivität aufweist oder der nicht die vollständige Aktivität des reifen Proteins aufweist.A signal peptide initiates (i) the insertion of the protein into the cell membrane, (ii) the translocation of the protein across the cell membrane, or (iii) the entry of the protein into the endoplasmic reticulum of the cell for secretion of the protein through the endoplasmic reticulum. Once the signal peptide has performed its task, it is usually removed by proteolytic cleavage to release a mature protein or a second precursor, called a "pro" precursor, which, like the first precursor, has no biological activity or does not have the full activity of the mature protein.
Ein "pro"-Fragment ist insofern nützlich, als es die Aktivität des Proteins blockiert oder modifiziert und so den Schutz der Zelle gegen die eventuellen toxischen Effekte des Proteins oder den Schutz des Proteins gegen eventuelle Modifikationen oder Abbaureaktionen ermöglicht. Es kann auch in einem bestimmten Umfang in den Sekretions-Mechanismus eingreifen. Am Ende des Sekretions-Prozesses wird das "pro" -Fragment des zweiten Vorläufers durch proteolytische Spaltung entfernt, um ein reifes Protein (in der aktiven Form) freizusetzen.A "pro" fragment is useful in that it blocks or modifies the activity of the protein, thus allowing the cell to be protected against the possible toxic effects of the protein or the protein to be protected against possible modifications or degradation reactions. It can also interfere to some extent with the secretion mechanism. At the end of the secretion process, the "pro" fragment of the second precursor is removed by proteolytic cleavage to release a mature protein (in the active form).
An der Verbindungsstelle zwischen dem "pre"-Fragment und dem "pro" -Fragment sowie an der Verbindungsstelle zwischen dem "pro"-Fragment und dem dem reifen Protein entsprechenden Fragment muß sich eine proteolytische Spaltungsstelle (Schnittstelle) befinden, die durch eine der Proteasen der Zelle erkannt wird, in der das Protein synthetisiert wird. Diese proteolytische Spaltungsstelle (Schnittstelle) besteht im allgemeinen aus einer Sequenz von zwei oder drei oder mehr Aminosäuren (nachstehend als "Proteolyse-Sequenz" bezeichnet), die für die Protease auf dem "pro"-Vorläufer zugänglich ist und die nicht zugänglich ist, wenn sie auf dem dem reifen Protein entsprechenden Fragment vorliegt. A priori sind drei Fälle möglich, die nachstehend unter Bezugnahme auf die Verbindung der Fragmente "pre" und "pro" erläutert werden:At the junction between the "pre" fragment and the "pro" fragment as well as at the junction between the "pro" fragment and the fragment corresponding to the mature protein, there must be a proteolytic cleavage site (cutting site) which is defined by one of the Proteases of the cell in which the protein is synthesized. This proteolytic cleavage site generally consists of a sequence of two or three or more amino acids (hereinafter referred to as the "proteolysis sequence") which is accessible to the protease on the "pro" precursor and which is not accessible when present on the fragment corresponding to the mature protein. A priori, three cases are possible, which are explained below with reference to the connection of the "pre" and "pro" fragments:
- die Protease schneidet am Kopf der Sequenz und infolgedessen muß die Proteolyse-Sequenz auf das "pro"-Fragment gelesen werden;- the protease cuts at the head of the sequence and consequently the proteolysis sequence must be read on the "pro" fragment;
- die Protease schneidet am Schwanz der Sequenz und infolgedessen muß die Proteoiyse-Sequenz auf das "pre"-Fragment gelesen werden;- the protease cuts at the tail of the sequence and consequently the proteolysis sequence must be read on the "pre" fragment;
- die Protease schneidet in der Mitte der Sequenz und infolgedessen muß die Proteolyse-Sequenz übergreifend auf die "pre"- und "pro"-Fragmente gelesen werden.- the protease cuts in the middle of the sequence and consequently the proteolysis sequence must be read across the "pre" and "pro" fragments.
Diese Betrachtungen gelten auch in ähnlicher Weise für die Spaltungsstelle (Schnittstelle), die an der Verbindung zwischen dem "pro"-Fragment und dem dem reifen Protein entsprechenden Fragment angeordnet ist.These considerations also apply in a similar way to the cleavage site (cutting site) located at the junction between the "pro" fragment and the fragment corresponding to the mature protein.
Für die Konstruktion von synthetischen Vorläufern unter Anwendung von gentechnischen Verfahren kann es erforderlich sein, natürliche Fragmente (d.h. solche, wie sie in der Natur zu finden sind) zu verwenden und an einer guten (geeigneten) Stelle eine neue proteolytische Spaltungsstelle (Schnittstelle) oder bestimmte Aminosäuren, um eine neue proteolytische Spaltungsstelle (Schnittstelle) zu erzeugen, in Assoziation mit den Fragmenten zu inserieren, wobei diese neue Spaltungsstelle (Schnittstelle) von einer der Proteasen der Zelle, in welcher der synthetische Vorläufer exprimiert wird, mit Sicherheit erkannt wird.For the construction of synthetic precursors using genetic engineering techniques, it may be necessary to use natural fragments (i.e. those found in nature) and to insert a new proteolytic cleavage site (cleavage site) or certain amino acids to create a new proteolytic cleavage site (cleavage site) in association with the fragments at a good (suitable) location, whereby this new cleavage site (cleavage site) is recognized with certainty by one of the proteases of the cell in which the synthetic precursor is expressed.
Ein Beispiel für diesen vorstehend beschriebenen Synthese-Typ und diese vor stehend beschriebene Art der Sekretion wird erläutert anhand des a-Sexual-Pheromons der Hefe S. cerevisiae, auch als Faktor a bezeichnet (der codiert wird durch das Gen MFal oder MFa2). Der Faktor α wird nämlich in Form des "pre-pro"-Vorläufers synthetisiert, wie von Kujan und Herskowitz in "Cell" (1982) 30:933, beschrieben. Die Aminosäuresequenz des Fragments "pre" des Vorläufers des Faktors α ist Met Arg Phe Pro 5er Ile Phe Thr Ala Val Leu Phe Ala Ala 5er 5er Ala Leu Ala, während diejenige des Fragments "pro" des Vorläufers des Faktors α ist Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala 5er Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Asp.An example of this type of synthesis and secretion described above is illustrated using the α-sexual pheromone of the yeast S. cerevisiae, also called factor a (which is encoded by the gene MFal or MFa2). Factor α is in fact synthesized in the form of the "pre-pro" precursor, as described by Kujan and Herskowitz in "Cell" (1982) 30:933. The amino acid sequence of the "pre" fragment of the precursor of factor α is Met Arg Phe Pro 5er Ile Phe Thr Ala Val Leu Phe Ala Ala 5er 5er Ala Leu Ala, while that of the "pro" fragment of the precursor of factor α is is Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala 5er Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Asp.
Überraschenderweise wurde jetzt gefunden, daß das N- terminale Ende des Vorläufers eines Hefe-Enzyms, dessen N- terminales Ende die folgende Aminosäuresequenz aufweist: Arg-Phe-Ser-Thr-Thr-Leu-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr-Ala-Ser-Gln-Val-Ser-Ala, als Signal-Peptid für die Sekretion von heterologen Proteinen verwendet werden kann ebenso wie verschiedene Varianten dieses N-terminalen Endes. Unter einem "heterologen Protein" versteht man ein Protein, das nicht in natürlicher Weise durch die Wirts-Zelle gebildet wird oder das durch eine Sequenz codiert wird, die nicht aus der Wirts-Zelle stammt.Surprisingly, it has now been found that the N-terminal end of the precursor of a yeast enzyme, whose N-terminal end has the following amino acid sequence: Arg-Phe-Ser-Thr-Thr-Leu-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr-Ala-Ser-Gln-Val-Ser-Ala, can be used as a signal peptide for the secretion of heterologous proteins, as can various variants of this N-terminal end. A "heterologous protein" is a protein that is not naturally produced by the host cell or that is encoded by a sequence that does not originate from the host cell.
Dementsprechend betrifft die vorliegende Erfindung ein isoliertes DNA-Fragment, das für ein Peptid codiert, dessen Aininosäuresequenz einen Grad der Homologie von mindestens 60 %, vorzugsweise von mindestens 80 %, mit den nachstehend angegebenen Aminosäureseguenzen (I) oder (II), vorzugsweise mit der Sequenz (II), aufweist:Accordingly, the present invention relates to an isolated DNA fragment encoding a peptide whose amino acid sequence has a degree of homology of at least 60%, preferably at least 80%, with the amino acid sequences (I) or (II) given below, preferably with the sequence (II):
(I) Arg-Phe-Ser-Thr-Thr-Leu-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-Ser-Gln.(I) Arg-Phe-Ser-Thr-Thr-Leu-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-Ser-Gln.
(II) Arg-Phe-Ser-Thr-Thr-Leu-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-Ser-Gln-Val-Ser-Ala.(II) Arg-Phe-Ser-Thr-Thr-Leu-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-Ser-Gln-Val-Ser-Ala.
Unter einem "isolierten DNA-Fragment" versteht man ein DNA-Fragment, dessen 3'-Ende nicht durch kovalente Bindung an ein DNA-Fragment gebunden ist, das für ein Enzym Codiert, das eine ß-1,3-Glucanase-Aktivität aufweist, wie insbesondere von Klebl & Tanner in "J. Bact", Nov. 1989, 171: 6259, beschrieben.An "isolated DNA fragment" is understood to mean a DNA fragment whose 3' end is not covalently linked to a DNA fragment encoding an enzyme having ß-1,3-glucanase activity, as described in particular by Klebl & Tanner in "J. Bact", Nov. 1989, 171: 6259.
Ein erfindungsgemäßes DNA-Fragment codiert insbesondere für ein Peptid, das die folgnede Aminosäuresequenz (III) aufweist:A DNA fragment according to the invention encodes in particular a peptide having the following amino acid sequence (III):
(III) R&sub1;-R&sub2;-R&sub3;-Thr-Thr-R&sub4;-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe- 1 5 10 Phe-Thr-Ala-R5-R&sub6; 15 15(III) R₁-R₂-R₃-Thr-Thr-R₄-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe- 1 5 10 Phe-Thr-Ala-R5-R₆ 15 15
worin bedeuten:where:
R&sub1; eine Aminosäure, ausgewählt aus Arg und Lys,R1 is an amino acid selected from Arg and Lys,
R&sub2; und R&sub6; jeweils eine Aminosäure, unabhängig voneinander ausgewählt ausR₂ and R₆ are each an amino acid, independently selected from
Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr und ValAla, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val
R&sub3; und R&sub5; jeweils eine Axninosäure, unabhängig voneinander ausgewählt aus Asp, Gly, Asn, Pro und 5er undR₃ and R₅ each represent an alkanoic acid independently selected from Asp, Gly, Asn, Pro and 5er and
R&sub4; eine Aminosäure, ausgewählt aus Val, Leu, Ala, Cys, Phe, Ile und Met.R4 is an amino acid selected from Val, Leu, Ala, Cys, Phe, Ile and Met.
Ein erfindungsgemäßes DNA-Fragment codiert vorzugsweise für eine Peptid, das die folgende Aminosäuresequenz (IV) aufweist:A DNA fragment according to the invention preferably encodes a peptide having the following amino acid sequence (IV):
(IV) R&sub1;-R&sub2;-R&sub3;-Thr-Thr-R&sub4;-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-R&sub5;-R&sub6;-R&sub7;(IV) R₁-R₂-R₃-Thr-Thr-R₄-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-R₅-R₆-R₆
worin bedeuten:where:
R&sub1; eine Aminosäure, ausgewählt aus Arg und Lys,R1 is an amino acid selected from Arg and Lys,
R&sub2; und R&sub6; jeweils eine Aminosäure, unabhängig voneinander ausgewählt ausR₂ and R₆ are each an amino acid, independently selected from
Ala, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr und ValAla, Asn, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val
R&sub3; und R&sub5; jeweils eine Aminosäure, unabhängig voneinander ausgewählt ausR₃ and R₅ each represent an amino acid, independently selected from
Asp, Gly, Asn, Pro und Ser,Asp, Gly, Asn, Pro and Ser,
R&sub4; eine Aminosäure, ausgewählt ausR4 is an amino acid selected from
Val, Leu Ala, Cys, Phe, Ile und Met,Val, Leu Ala, Cys, Phe, Ile and Met,
R&sub7; eine Proteolyse-Sequenz.R7 is a proteolysis sequence.
R&sub7; ist vorzugsweise eine Proteolyse-Sequenz R&sub8;-R&sub9;--R�7 is preferably a proteolysis sequence R�8-R�9--
R&sub1;&sub0;, in der bedeuten:R₁₀, in which:
R&sub8; eine Aminosäure, ausgewählt ausR�8 is an amino acid selected from
Ala, Val, Ser, Cys, Gly, Ile, Leu und Thr,Ala, Val, Ser, Cys, Gly, Ile, Leu and Thr,
R&sub9; eine Aminosäure, ausgewählt ausR�9 is an amino acid selected from
Ala, Arg, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr und Val undAla, Arg, Cys, Gln, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val and
R&sub1;&sub0; eine Aminosäure, ausgewählt ausR₁₀ is an amino acid selected from
Ala, Cys, Gly, Leu, Pro, Gln, Ser und Thr.Ala, Cys, Gly, Leu, Pro, Gln, Ser and Thr.
Erfindungsgemäß codiert ein DNA-Fragment vorzugsweise für ein Peptid, das eine Aminosäuresequenz aufweist, die ausgewählt wird unter den folgenden Aminosäuresequenzen (V) (VI) , (VII) und (VIII):According to the invention, a DNA fragment preferably encodes a peptide having an amino acid sequence that is selected from the following amino acid sequences (V) (VI), (VII) and (VIII):
(V) Arg-Phe-Ser-Thr-Thr-Leu-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-Ser-Gln(V) Arg-Phe-Ser-Thr-Thr-Leu-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-Ser-Gln
(VI) Arg-Phe-Ser-Thr-Thr-Val-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-Scr-Gln(VI) Arg-Phe-Ser-Thr-Thr-Val-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe-Thr- Ala-Scr-Gln
(VII) Arg-Phe-Ser-Thr-Thr-Leu-Ala-Ala-Thr-Ala-Leu-Phe-Phe- Thr-Ala-Ser-Gln-R&sub7;(VII) Arg-Phe-Ser-Thr-Thr-Leu-Ala-Ala-Thr-Ala-Leu-Phe-Phe- Thr-Ala-Ser-Gln-R�7;
worin R&sub7; wie oben definiert ist,wherein R�7 is as defined above,
(VIII) Arg-Phe-Ser-Thr-Thr-Val-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe- Thr-Ala-Ser-Gln-R&sub7;(VIII) Arg-Phe-Ser-Thr-Thr-Val-Ala-Thr-Ala-Ala-Thr-Ala-Leu-Phe-Phe- Thr-Ala-Ser-Gln-R�sub7;
worin R&sub7; wie oben definiert ist.wherein R7 is as defined above.
R&sub7; steht besonders bevorzugt für eine Sequenz, in der R&sub8; Val, R&sub9; Ser und R&sub1;&sub0; Ala bedeuten.R₇ particularly preferably represents a sequence in which R₈ is Val, R₇ is Ser and R₁₀ is Ala.
Ein erfindungsgemäßes DNA-Fragment weist als Nucleotid-Sequenz ganz besonders bevorzugt eine der folgenden Sequenzen (IX), (X), (XI) und (XII) auf: A DNA fragment according to the invention most preferably has one of the following sequences (IX), (X), (XI) and (XII) as nucleotide sequence:
Die durch die erfindungsgemäßen DNA-Fragmente codierten Peptide enthalten eine hydrophobe Region, die eine α- Helix-Struktur aufweist, zwischen der Aminosäure in der Position 3 und der Aminosäure in der Position 18. Diese Struktur trägt dazu bei, sie für eine Verwendung als Signal-Peptid geeignet zu machen. Es ist bekannt, daß der hydrophobe Abschnitt der Signal-Sequenzen überwiegend aus den Aminosäuren Ala, Cys, Phe, Ile, Leu, Met, Val besteht. Man kann auch bestimmte Modifikationen von Aminosäuren vorsehen, die keine Veränderung der Fähigkeit des Signal-Peptids, seine Rolle zu spielen, hervorrufen.The peptides encoded by the DNA fragments of the invention contain a hydrophobic region having an α-helix structure between the amino acid in position 3 and the amino acid in position 18. This structure helps to make them suitable for use as a signal peptide. It is known that the hydrophobic portion of the signal sequences consists predominantly of the amino acids Ala, Cys, Phe, Ile, Leu, Met, Val. It is also possible to provide certain modifications of amino acids which do not cause any alteration in the ability of the signal peptide to play its role.
Unter einem anderen Aspekt betrifft die vorliegende Erfindung infolgedessen die Verwendung eines erfindungsgemäßen DNA-Fragments als DNA-Fragment, das für ein Signal-Peptid codiert, das geeignet ist für die Sekretion eines heterologen Proteins durch eine Wirtszelle, in der das heterologe Protein synthetisiert wird. Allgemein kann die Erfindung durchgeführt werden in einer Prokaryontenzelle oder in einer Eukaryontenzelle, vorzugsweise in letzterer. Die Eukaryontenzelle kann beispielsweise eine Säugetierzelle oder eine Hefezelle sein. Die Sekretion eines heterologen Proteins mit Hilfe eines Signal-Peptids, das durch ein erfindungsgemäßes Fragment codiert wird, wird vorzugsweise durchgeführt durch eine Hefezelle, beispielsweise des Genus Saccharomyces, insbesondere der Species S. cerevisiae.In another aspect, the present invention therefore relates to the use of a DNA fragment according to the invention as a DNA fragment encoding a signal peptide suitable for the secretion of a heterologous protein by a host cell in which the heterologous protein is synthesized. In general, the invention can be carried out in a prokaryotic cell or in a eukaryotic cell, preferably in the latter. The eukaryotic cell can be, for example, a mammalian cell or a yeast cell. The secretion of a heterologous protein with the aid of a signal peptide encoded by a fragment according to the invention is preferably carried out by a yeast cell, for example of the genus Saccharomyces, in particular the species S. cerevisiae.
Sofern die DNA-Fragmente für ein Signal-Peptid codieren, geht den erfindungsgemäßen DNA-Fragmenten selbstverständlich ein Translations-Initiierungs-codon voraus, im allgemeinen ein Codon, der für ein Methionin codiert, insbesondere ein ATG.If the DNA fragments code for a signal peptide, the DNA fragments according to the invention are of course preceded by a translation initiation codon, generally a codon that codes for a methionine, in particular an ATG.
Die erfindungsgemäßen DNA-Fragmente können für die Konstruktion einer Kassette zur Expression eines Proteins verwendet werden, denen der Initiierungs-codon allein oder in Kombination mit anderen Bestandteilen, beispielsweise einem Fragment "pro", vorausgeht. Diese DNA-Fragmente können durch chemische Synthese mittels eines Oligonucleotid- Synthetisators nach einem dem Fachmann allgemein bekannten Verfahren hergestellt werden.The DNA fragments according to the invention can be used for the construction of a cassette for the expression of a protein, which is preceded by the initiation codon alone or in combination with other components, for example a "pro" fragment. These DNA fragments can be produced by chemical synthesis using an oligonucleotide synthesizer according to a method generally known to those skilled in the art.
Die Erfindung betrifft außerdem eine Kassette zur Expression eines heterologen Proteins, die umfaßt ein erfindungsgemäßes DNA-Fragment als DNA-Fragment, das für das Signal-Peptid des genannten heterologenen Proteins Codiert. Im einzelnen umfaßt die erfindungsgemäße Expressionskassette, welche die für die Sekretion eines reifen heterologen Proteins erforderliche Information enthält, in aufeinanderfolgender Weise mindestens:The invention also relates to a cassette for the expression of a heterologous protein, which comprises a DNA fragment according to the invention as a DNA fragment encoding the signal peptide of said heterologous protein. In particular, the expression cassette according to the invention, which contains the information required for the secretion of a mature heterologous protein, comprises in succession at least:
a) ein DNA-Fragment, das Transkriptions- und Translations-Initiierungssignale aufweist,a) a DNA fragment containing transcription and translation initiation signals,
b) ein erfindungsgemäßes DNA-Fragment,b) a DNA fragment according to the invention,
c) ein DNA-Fragment, das für ein reifes heterologes proteins codiert (mit einem Translations-Schlußcodon).c) a DNA fragment encoding a mature heterologous protein (with a translation termination codon).
Bei einer ersten Variante kann man das Fragment (b) in Phase mit dem Fragment (c) direkt fusionieren, sofern an der Verbindungsstelle zwischen dem Fragment (b) und dem Fragment (c) eine DNA-Sequenz vorliegt, die für eine proteolytische Spaltungsstelle (Schnittstelle) codiert, um die Freisetzung eines reifen Proteins am Ende des Expressions- und Sekretions-Prozesses zu erlauben.In a first variant, fragment (b) can be directly fused in phase with fragment (c), provided that at the junction between fragment (b) and fragment (c) there is a DNA sequence encoding a proteolytic cleavage site (cutting site) to allow the release of a mature protein at the end of the expression and secretion process.
Vorzugsweise wird die DNA-Sequenz, die für eine proteolytische Spaltungsstelle (Schnittstelle) codiert, auf das Fragment (b) gelesen.Preferably, the DNA sequence encoding a proteolytic cleavage site (cleavage site) is read onto fragment (b).
Bei einer zweiten Variante umfaßt eine erfindungsgemäße Expressionskassette außerdem ein DNA-Fragment (b'), das für eine Peptid-Fragment "pro" codiert. Dieses Fragment (b') wird in Phase mit dem Fragment (b) und dem Fragment (c) fusioniert, an der Verbindungsstelle zwischen den Fragmenten (b) und (b') einerseits und den Fragmenten (b') und (c) andererseits eine DNA-Sequenz vorhanden ist, die für eine proteolytische Spaltungsstelle (Schnittstelle) codiert.In a second variant, an expression cassette according to the invention also comprises a DNA fragment (b') that codes for a peptide fragment "pro". This fragment (b') is fused in phase with the fragment (b) and the fragment (c), at the connection point between the fragments (b) and (b') on the one hand and the fragments (b') and (c) on the other hand there is a DNA sequence that codes for a proteolytic cleavage site (cleavage site).
In den erfindungsgemäßen Kassetten können zahlreiche Fragmente (b') verwendet werden. Insbesondere können verschiedene Fragmente (b') auf synthetische Weise konstruiert werden. Als Beispiel ist nachstehend die Konstruktion eines synthetischen Fragments (b') beschrieben, bei dem man von der DNA-Sequenz ausgeht, die für das Fragment "pro" des Vorläufers des Faktors α codiert. Diese Sequenz kann insgesamt oder zum Teil verwendet werden. Bei einer speziellen Ausführungsform verwendet man die Sequenz, die deletiert worden ist aus dem Abschnitt, der für die Aminosäure codiert in der Position 3 bis 42, auf dem Fragment "pro", d.h. die Sequenz, die codiert für Ala Pro Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Asp. Zur Bildung des Fragments (b') folgt auf diese zuletzt genannte DNA-Sequenz eine Sequenz, die codiert für (i) ein Peptid, das eine proteolytische Spaltungsstelle (Schnittstelle) umfaßt, oder (ii) eine proteolytische Spaltungsstelle (Schnittstelle), wobei die zuletzt genannte Ausführungsform bevorzugt ist. Bei der zuletzt genannten Spaltungsstelle (Schnittstelle) handelt es sich insbesondere um Lys-Arg oder Arg-Arg, die durch die Hefe-Endopeptidase yscF erkannt wird, die codiert wird durch das Gen KEX2 und die im Bereich des C-terminalen Endes des Dipeptids Lys-Arg oder Arg-Arg schneidet. Zusammenfassend codiert ein Fragment (b') insbesondere für Ala Pro Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Asp Lys Arg.Numerous fragments (b') can be used in the cassettes according to the invention. In particular, various fragments (b') can be constructed synthetically. As an example, the construction of a synthetic fragment (b') starting from the DNA sequence coding for the "pro" fragment of the precursor of factor α is described below. This sequence can be used in whole or in part. In a specific embodiment, the sequence that has been deleted from the section coding for the amino acid in position 3 to 42 on the fragment "pro", i.e. the sequence coding for Ala Pro Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Asp, is used. To form the fragment (b'), this last-mentioned DNA sequence is followed by a sequence coding for (i) a peptide comprising a proteolytic cleavage site (cleavage site), or (ii) a proteolytic cleavage site (cleavage site), the last-mentioned embodiment being preferred. The last-mentioned cleavage site (cutting site) is in particular Lys-Arg or Arg-Arg, which is recognized by the yeast endopeptidase yscF, which is encoded by the KEX2 gene and which cuts in the region of the C-terminal end of the dipeptide Lys-Arg or Arg-Arg. In summary, a fragment (b') encodes in particular Ala Pro Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Asp Lys Arg.
Die Sequenz (a) umfaßt insbesondere einen funktionellen Promotor in der Zelle, in der man das heterologe Protein, das durch das Fragment (c) codiert wird, zu synthetisieren wünscht, vorzugsweise einen funktionellen Promotor bei der Hefe. Beispielhaft können Hefe aufbauende Promotoren verwendet werden, deren Funktionalität durch die Transkription von Genen bestätigt wurde, die für heterologe Proteine codieren, beispielsweise die Promotoren PGK, ENO1, MFα1 oder auch induzierbare Promotoren wie PH05, GAL1. Wenn man in der Expressionskassette Elemente des Gens MFα1 von Hefe verwendet, kann man beispielsweise den Promotor des Gens MFα1 verwenden.The sequence (a) comprises in particular a functional promoter in the cell in which it is desired to synthesize the heterologous protein encoded by the fragment (c), preferably a functional promoter in yeast. For example, yeast-based promoters can be used whose functionality has been confirmed by the transcription of genes encoding heterologous proteins, for example the PGK, ENO1, MFα1 promoters or inducible promoters such as PH05, GAL1. If elements of the yeast MFα1 gene are used in the expression cassette, the promoter of the MFα1 gene can be used, for example.
Schließlich können die Expressionskassetten auch ein DNA-Fragment (d) umfassen, das Signale zur Beendigung der Transkription, vorzugsweise funktionelle Signale bei der Hefe, beispielsweise dasjenige des Gens PGK, umfaßt.Finally, the expression cassettes can also comprise a DNA fragment (d) which comprises signals for the termination of transcription, preferably functional signals in yeast, for example that of the PGK gene.
Allgemein kann eine erfindungsgemäße Expressionskassette in eine Prokaryontenzelle oder in eine Eukaryontenzelle, vorzugsweise in eine Eukaryontenzelle, z.B. in eine Säugetierzelle oder Hefezelle, eingeführt werden, wobei eine Hefezelle ganz besonders bevorzugt ist. Diese Einführung kann in der Weise erfolgen, daß man die Kassette in einem autonomen Replikations-Plasmid oder in einer Konstruktion, die für die Integration für die direkte Einführung in das Genom der Hefe bestimmt ist, so anordnet, daß man in beiden Fällen eine transformierte Zelle erhält.In general, an expression cassette according to the invention can be introduced into a prokaryotic cell or into a eukaryotic cell, preferably into a eukaryotic cell, e.g. into a mammalian cell or yeast cell, with a yeast cell being particularly preferred. This introduction can be carried out by arranging the cassette in an autonomous replication plasmid or in a construction intended for integration for direct introduction into the genome of the yeast, so that in both cases a transformed cell is obtained.
Wenn das Plasmid autonom ist, weist es Elemente auf, die seine Teilung und seine Replikation gewährleisten, beispielsweise einen Replikationsursprung wie denjenigen des Hefe-Plasmids 2 u. Außerdem kann das Plasmid Selektions-Elemente, z.B. das Gen URA3 oder LEU2 umfassen, welche die Komplementierung von Hefe ura3 oder leu2 gewährleisten. Insbesondere kann man mit Vorteil das Gen URA3 verwenden, das aus seinem Promotor deletiert worden ist (URA3-d).If the plasmid is autonomous, it contains elements that ensure its division and replication, for example an origin of replication such as that of yeast plasmid 2u. In addition, the plasmid may contain selection elements, such as the URA3 or LEU2 gene, which ensure the complementation of yeast ura3 or leu2. In particular, it is advantageous to use the URA3 gene deleted from its promoter (URA3-d).
Diese Plasmide können auch Elemente umfassen, die ihre Replikation in Bakterien gewährleisten, wenn das Plasmid ein Shuttle-Plasmid (Schaukel-Plasmid) sein muß, beispielsweise einen Replikationsursprung, wie denjenigen von pBR322, ein Selektions-Marker-Gen wie Amp und/oder andere Elemente, die dem Fachmann bekannt sind.These plasmids may also include elements that ensure their replication in bacteria when the Plasmid must be a shuttle plasmid, for example an origin of replication such as that of pBR322, a selection marker gene such as Amp and/or other elements known to the person skilled in the art.
In Übereinstimmung mit den vorstehenden Angaben betrifft die vorliegende Erfindung außerdem eine Zelle, die transformiert worden ist durch (i) ein erfindungsgemäßes DNA-Fragment oder (ii) eine erfindungsgemäße Expressions-Kassette, die entweder in ein Plasmid inseriert oder in das Genom der Zelle integriert worden ist.In accordance with the above, the present invention also relates to a cell which has been transformed by (i) a DNA fragment according to the invention or (ii) an expression cassette according to the invention which has either been inserted into a plasmid or integrated into the genome of the cell.
Wenn der Promotor derjenige des Gens MFα1 ist, ist die transformierte Hefezelle vorzugsweise eine solche vom Sexual-Typ MATα. Man verwendet beispielsweise einen Stamm vom Genotyp ura3 oder leu2 oder einen anderen, komplementiert durch das Plasmid, um die Aufrechterhalung des Plasmids in der Hefe durch einen geeigneten Selektionsdruck zu gewährleisten.When the promoter is that of the MFα1 gene, the transformed yeast cell is preferably of the sexual type MATα. For example, a strain of the genotype ura3 or leu2 or another is used, complemented by the plasmid in order to ensure the maintenance of the plasmid in the yeast by an appropriate selection pressure.
Schließlich betrifft die vorliegende Erfindung ein Verfahren zur Herstellung eines heterologen Proteins, das dadurch gekennzeichnet ist, daß man eine erfindungsgemäße Zelle kultiviert und daß man das genannte Protein aus dem Kulturmedium gewinnt (abtrennt). Dieses Verfahren ist anwendbar auf die Herstellung jedes Proteins heterologer Art. Unter diesen Proteinen können insbesondere genannt werden das Hirudin oder Defensine, beispielsweise das Defensin A.Finally, the present invention relates to a process for producing a heterologous protein, which is characterized in that a cell according to the invention is cultivated and in that said protein is recovered (separated) from the culture medium. This process is applicable to the production of any protein of a heterologous nature. Among these proteins, mention may be made in particular of hirudin or defensins, for example defensin A.
Die Erfindung betrifft insbesondere ein Verfahren zur Sekretion von Hirudin in reifer Form, bei dein man von Hefestämmen ausgeht, die erfindungsgemäß transformiert worden sind.The invention relates in particular to a process for the secretion of hirudin in mature form, starting from yeast strains which have been transformed according to the invention.
Die Erfindung ist insbesondere anwendbar auf die Herstellung von Hirudin, weshalb eines der erläuternden erfindungsgemäßen Beispiele dieses Protein betrifft. Das Hirudin, dessen Hauptquelle in den Speicheldrüsen von medizinischen Blutegeln zu finden ist, ist nämlich ein sehr spezifischer und sehr wirksamer Thrombin-Inhibitor. Es handelt sich dabei um ein sehr interessantes therapeutisches Agens, dessen klinische Verwendung eine sehr große Reinheit des Produkts erfordert und das daher ein interessanter Kandidat für die Herstellung auf gentechnischem Wege ist.The invention is particularly applicable to the production of hirudin, which is why one of the illustrative examples of the invention concerns this protein. Hirudin, whose main source is found in the salivary glands of medicinal leeches, is in fact a very specific and very effective thrombin inhibitor. It is a very interesting therapeutic Agent whose clinical use requires a very high purity of the product and which is therefore an interesting candidate for production by genetic engineering.
Eine bestimmte Anzahl von natürlichen Varianten des Hirudins wurden bereits identifiziert und sie werden bezeichnet als HV1, HV2, HV3. Nachstehend werden diese natürlichen Varianten sowie weitere Analoga auf gentechnischein Wege in verschiedenen Wirtszellen hergestellt, wie beispielsweise in den europäischen Patentpublikationen EP- A-0200655, EP-A-0273800 der Anmelderin beschrieben. Ein Vergleich zwischen dem durch Escherichia coli (E. coli) synthetisierten Hirudin und dem durch eine Hefe des Genus S. cerevisiae synthetisierten Hirudin hat gezeigt, daß das durch E. coli synthetisierte Hirudin intrazellulär bleibt und somit gereinigt werden muß, wobei man von einer sehr großen Anzahl von Polypeptiden von E. coli ausgeht. Es ist daher besonders vorteilhaft (interessant), ein Gen von Hirudin in der Hefe so exprimieren zu können, daß man ein in reifer Form sekretiertes Hirudin erhält, ohne daß die Hefe für den Menschen pyrogene oder toxische Substanzen bildet.A certain number of natural variants of hirudin have already been identified and are designated HV1, HV2, HV3. These natural variants and other analogues are produced below by genetic engineering in various host cells, as described for example in the applicant's European patent publications EP-A-0200655, EP-A-0273800. A comparison between hirudin synthesized by Escherichia coli (E. coli) and hirudin synthesized by a yeast of the genus S. cerevisiae has shown that hirudin synthesized by E. coli remains intracellular and thus has to be purified, starting from a very large number of E. coli polypeptides. It is therefore particularly advantageous (interesting) to be able to express a hirudin gene in yeast in such a way that a hirudin secreted in mature form is obtained without the yeast producing pyrogenic or toxic substances for humans.
Die Erfindung ist anwendbar auf alle Hirudinmoleküle, d.h. auf alle natürlichen Varianten des Hirudins als solche oder auf solche, die eine oder mehrere Mutationen durchgemacht haben unter Beibehaltung ihrer Antithrombose-Aktivität, wobei der zuletzt genannte Varianten-Typ als Analogen bezeichnet wird. Die nachstehenden Beispiele betreffen insbesondere das Analogon, das als rHV2Lys47 bezeichnet wird (für die rekombinante Variante HV2, die eine Mutation der Aminosäurese Asp in der Position 47 zur Aminosäure Lys durchgemacht hat), wie in der bereits genannten europäischen Patentpublikation EP-A-0273800 beschrieben.The invention is applicable to all hirudin molecules, i.e. to all natural variants of hirudin as such or to those which have undergone one or more mutations while retaining their antithrombotic activity, the latter type of variant being referred to as analogues. The examples below relate in particular to the analogue referred to as rHV2Lys47 (for the recombinant variant HV2 which has undergone a mutation of the amino acid Asp at position 47 to the amino acid Lys), as described in the aforementioned European patent publication EP-A-0273800.
Die vorliegende Erfindung ist auch auf die Herstellung von Defensinen anwendbar. Die Defensine, auch als Phormicine bezeichnet, sind Peptide, die ursprünglich aus der Hämolyinphe bestiminter Insekten, der Diptera (Zweiflügler), extrahiert wurden, die eine bacterizide Aktivität gegenüber Gram-positiven Keimen aufweisen. Diese Defensine sind in der europäischen Patentanmeldung EP-A- 5349 451 ausführlich beschrieben. Das Defensin A ist ein basisches Peptid, das die Sequenz Ala Thr Cys Asp Leu Leu Ser Gly Thr Gly Ile Asn His Ser Ala Cys Ala Ala His Cys Leu Leu Arg Gly Asn Arg Gly Gly Tyr Cys Asn Gly Lys Gly Val Cys Val Cys Arg AsnThe present invention is also applicable to the production of defensins. Defensins, also called phormicins, are peptides originally derived from from the haemolymph of certain insects, the Diptera (Two-winged insects), which have a bactericidal activity against Gram-positive germs. These defensins are described in detail in the European patent application EP-A- 5349 451. Defensin A is a basic peptide that has the sequence Ala Thr Cys Asp Leu Leu Ser Gly Thr Gly Ile Asn His Ser Ala Cys Ala Ala His Cys Leu Leu Arg Gly Asn Arg Gly Gly Tyr Cys Asn Gly Lys Gly Val Cys Val Cys Arg Asn
aufweist. Das Defensin B unterscheidet sich von dem Defensin A nur durch die Aminosäure in der Position 32, in der ein Arginin ein Glycin ersetzt.Defensin B differs from defensin A only by the amino acid in position 32, in which an arginine replaces a glycine.
Die nachfolgenden Beispiele zeigen weitere Charakteristika und Vorteile der vorliegenden Erfindung. Diese Beispiele werden durch die folgenden Figuren erläutert:The following examples show further characteristics and advantages of the present invention. These examples are illustrated by the following figures:
- Die Fig. 1 zeigt die schematische Struktur des Plasmids pTG2958.- Figure 1 shows the schematic structure of the plasmid pTG2958.
- Die Fig. 2 zeigt die schematische Struktur der Vektoren M13TG3839 und M13TG3841. Für M13TG3839 entsprechen die schraffierten Rechtecke an den Enden dem M13TG103 und für M13TG3841 entsprechen sie dem M13TG3149.- Fig. 2 shows the schematic structure of the vectors M13TG3839 and M13TG3841. For M13TG3839, the hatched rectangles at the ends correspond to M13TG103 and for M13TG3841 they correspond to M13TG3149.
- Die Fig. 3 zeigt die schematische Struktur des Vektors M13TG3845, wobei das schraffierte Rechteck dem M13TG3149 entspricht.- Figure 3 shows the schematic structure of the vector M13TG3845, where the hatched rectangle corresponds to the M13TG3149.
- Die Fig. 4 zeigt die schematische Struktur des Plasmids pTG3828.- Figure 4 shows the schematic structure of the plasmid pTG3828.
- Die Fig. 5 zeigt die schematische Struktur des Plasmids pTG3864.- Figure 5 shows the schematic structure of the plasmid pTG3864.
Das Plasmid pTG2958 (Fig. 1) unterscheidet sich kaum von dem Plasmid pTG1833, das in der europäischen Patentpublikation EP-A-252854 beschrieben ist, welches die für rHV2Asp47 codierende Sequenz trägt. Das Plasmid pTG2958 enthält nicht die künstlich eingeführte Restriktionsstelle HindIII. Das Plasmid pTG2958 enthält:Plasmid pTG2958 (Fig. 1) differs little from plasmid pTG1833 described in European patent publication EP-A-252854, which carries the coding sequence for rHV2Asp47. Plasmid pTG2958 does not contain the artificially introduced restriction site HindIII. The plasmid pTG2958 contains:
- ein Fragment von 1217 Basenpaaren, das der 5'-Region des Gens MFα1 entspricht (das enthält den Promotor, die für das Signal-Peptid codierende Sequenz, die Region "pro" und einen für das Peptid Lys-Arg codierende Sequenz) und von 4 Basenpaaren (Stelle BglIIº, Klenow-Behandlung),- a fragment of 1217 base pairs corresponding to the 5' region of the MFα1 gene (containing the promoter, the signal peptide coding sequence, the "pro" region and a sequence coding for the Lys-Arg peptide) and of 4 base pairs (BglIIº site, Klenow treatment),
- ein Fragment von 234 Basenpaaren, das die komplementäre DNA von rHV2Lys47 enthält,- a fragment of 234 base pairs containing the complementary DNA of rHV2Lys47,
- ein Fragment von 243 Basenpaaren, das den Hefe-Terminator PGK umfaßt,- a fragment of 243 base pairs comprising the yeast terminator PGK,
- das Fragment PvuII-EcoRI von pBR322, das unter anderen den Replikationsursprung dieses Plasmids und das Gen für die Resistenz gegen Ampicillin (2292 Basenpaare) umfaßt,- the PvuII-EcoRI fragment of pBR322, which includes, among other things, the origin of replication of this plasmid and the gene for resistance to ampicillin (2292 base pairs),
- das Fragment EcoRI-HindIII des Plasmids 2 u der Hefe (Form B), welches das Hefe-Gen LEU2 in der deletierten Form enthält, das in die Stelle PstI inseriert worden ist,- the EcoRI-HindIII fragment of yeast plasmid 2u (form B) containing the yeast gene LEU2 in the deleted form inserted into the PstI site,
- ein Fragment HindIII-Smal des Hefe-Gens URA3.- a fragment HindIII-Smal of the yeast gene URA3.
Das Fragment NcoI-Ncol des Vektors pTG2958, das die Sequenzen LEU2-d, 2 u und URA3 trägt, wird ersetzt durch das Fragment NcoI-NcoI von pTG2800, wie in der europäischen Patentpublikation EP-A-0268501 beschrieben, welches die Sequenzen des Plasmids 2 u und des Gens URA3, das aus seinem Promotor deletiert worden ist (URA3-d), zur Bildung von pTG2877.The NcoI-NcoI fragment of the vector pTG2958 carrying the sequences LEU2-d, 2 u and URA3 is replaced by the NcoI-NcoI fragment of pTG2800 as described in the European patent publication EP-A-0268501, which contains the sequences of the plasmid 2 u and the gene URA3 deleted from its promoter (URA3-d) to form pTG2877.
Der Vektor M13TG3839 (Fig. 2) leitet sich ab von M13TG103 abgeleitet ist (M.P. Kieny et al. (1983) "Gene", 26, 91-99), in welches das Fragment HindIII-HindIII von pTG2877 in die gleiche Stelle eingeführt worden ist. Eine Restriktionsstelle SalI wird in diesen Vektor stromabwärts von dem Translationsterminierungs-Codon der Region, die für rHV2Lys47 codiert, durch gerichtete Mutagenese mit Hilfe des folgenden Oligonucleotids eingeführt:The vector M13TG3839 (Fig. 2) is derived from M13TG103 (M.P. Kieny et al. (1983) "Gene", 26, 91-99) in which the fragment HindIII-HindIII of pTG2877 has been introduced into the same site. A restriction site SalI is introduced into this vector downstream of the translation termination codon of the region encoding rHV2Lys47 by site-directed mutagenesis using the following oligonucleotide:
5' CAATGAAAAATGGTCGACTATCAATCATAG5' CAATGAAAAATGGTCGACTATCAATCATAG
unter Bildung von M13TG3839 SalI. Eine Restriktionsstelle SphI wird dann stromaufwärts von der Expressionskassette eingeführt unter Eliminierung der Sequenz URA3-d durch gerichtete Mutagenese mit Hilfe des folgenden Oligonucleotids:to form M13TG3839 SalI. A restriction site SphI is then placed upstream of the expression cassette introduced by eliminating the sequence URA3-d by site-directed mutagenesis using the following oligonucleotide:
5' GACGGCCAGTGAATTGGCATGCTATTGATAAGATTTAAAG unter Bildung von M13TG3840.5' GACGGCCAGTGAATTGGCATGCTATTGATAAGATTTAAAG to form M13TG3840.
Der Vektor M13TG131 (M.P. Kieny et al. (1983), "Gene", 26, 91-99) wird durch PstI gespalten, die Enden werden durch Behandlung mit dem Klenow-Fragment der Polymerase-DNA I freigelegt, um anschließend mit sich selbst religiert zu werden unter Bildung von M13TG3160. Dieser Vektor wird anschließend durch SmaI und EcoRV gespalten und dann wieder religiert unter Bildung von M13TG3149.The vector M13TG131 (M.P. Kieny et al. (1983), "Gene", 26, 91-99) is cleaved by PstI, the ends are exposed by treatment with the Klenow fragment of polymerase DNA I, and then religated to itself to form M13TG3160. This vector is then cleaved by SmaI and EcoRV and then religated again to form M13TG3149.
Das Fragment SphI-SaII von M13TG3840 (wie vorstehend beschrieben), welche die Kassette zur Expression von rHV2Lys47 (ohne Transkriptions-Terminierungs-Sequenz) trägt, wird in die Stelle SphI-SalI von M13TG3149 eingeführt unter Bildung von M13TG3841 (Fig. 2).The SphI-SalI fragment of M13TG3840 (as described above), carrying the cassette for expression of rHV2Lys47 (without transcription termination sequence), is inserted into the SphI-SalI site of M13TG3149 to form M13TG3841 (Fig. 2).
Die für die Aminosäuren in der Position 3 bis 42 an dem Fragment "pro" des Vorläufers von MFα1 codierende Sequenz wird aus M13TG3149 eliminiert durch gerichtete Mutagenese und eine Restriktionsstelle SmaI, die unter Verwendung des folgenden Oligonucleotids eingeführt wird:The sequence coding for the amino acids in positions 3 to 42 on the "pro" fragment of the MFα1 precursor is eliminated from M13TG3149 by site-directed mutagenesis and a SmaI restriction site is introduced using the following oligonucleotide:
5' GTCCGCATTAGCTGCTCCCGGGTTATTGTTTATAAAT5' GTCCGCATTAGCTGCTCCCCGGGTTATTGTTTATAAAT
unter Bildung dessen, was nachfolgend als deletierte "pro"-Sequenz bezeichnet wird. Man erhält so M13TG3842. Eine Restriktionsstelle BamHI, die das ATG des Vorläufers des Faktorens α zerstört, wird ohne diesen Vektor durch gerichtete Mutagenese mit dem folgenden Oligonucleotid eingeführt:to form what is referred to below as the deleted "pro" sequence. M13TG3842 is thus obtained. A BamHI restriction site, which destroys the ATG of the precursor of factor α, is introduced without this vector by directed mutagenesis with the following oligonucleotide:
5' AATATAAACGATTAAAAGGATCCGATTTCCTTCAATTTTTA5' AATATAAACGATTAAAAGGATCCGATTTCCTTCAATTTTTA
Man erhält so M13TG3843. Nach der Phosphorylierung werden die folgenden oligonucleotideThis gives M13TG3843. After phosphorylation, the following oligonucleotides
5' GATCCGTTTCTCTACTACAGTCGCTACTGCAGCTACTGCGCTATT GCAAAGTGATGATGTCAGCGATG 5'5' GATCCGTTTCTCTACTACAGTCGCTACTGCAGCTACTGCGCTATT GCAAAGTGATGATGTCAGCGATG 5'
undand
5' TTTCACAGCCTCCCAAGTTTCAGCTGCTCCC ACGTCGATGACGCGATAAAAAGTGTCGGAGGGTTCAAAGTCGACGAGGG 5'5' TTTCACAGCCTCCAAGTTTCAGCTGCTCCC ACGTCGATGACGCGATAAAAAGTGTCGGAGGGTTCAAAGTCGACGAGGG 5'
in den Vektor M13TG3843 inseriert, mit BamHI und SmaI geschnitten, um auf diese Weise die Sequenz XI ohne ATG einzuführen. Zur Wiederherstellung von ATG wird die Stelle BamHI durch gerichtete Mutagenese unter Verwendung des folgenden Oligonucleotids eliminiert:inserted into the vector M13TG3843, cut with BamHI and SmaI to introduce the sequence XI without ATG. To restore ATG, the BamHI site is eliminated by site-directed mutagenesis using the following oligonucleotide:
5' AATATAAACGATTAAAAGAATGCGTTTCTCTACTACAGTC5' AATATAAACGATTAAAAGAATGCGTTTCTCTACTACAGTC
unter Bildung des Vektors MI3TG3845 (Fig. 3), der umfaßt:forming the vector MI3TG3845 (Fig. 3), which comprises:
- den Promotor des Gens MFα1, gefolgt von einem Codon ATG als Fragment (a),- the promoter of the gene MFα1, followed by a codon ATG as a fragment (a),
- die Sequenz XI als Fragment (b)- the sequence XI as fragment (b)
- die Sequenz "pro", die aus dem Gen MFα1 deletiert worden ist, gefolgt von codons, die für Lys-Arg codieren, als Fragment (b'),- the sequence "pro" deleted from the gene MFα1, followed by codons coding for Lys-Arg, as fragment (b'),
- die Sequenz, die für rHV2Lys47 codiert, als Fragment (c), und- the sequence encoding rHV2Lys47 as a fragment (c), and
- einen Teil des Vektors M13TG3149.- part of the vector M13TG3149.
Das in der europäischen Patentpublikation EP-A- 0252854 beschriebene Plasmid pTG848 wird mit BglII digeriert, dann religiert unter Bildung von pTG2886. Das große Fragment HindIII-EcoRI von pTG2886 wird in Gegenwart der Ligase T4 mit dem Fragment HindIII-EcoRI von 2,1 kb des Plasmids pFL1 ligiert (S.A. Parent et al. (1985), "Yeast", 1, 83-138), welches die Sequenz des Plasmids 2 u von S. cerevisiae trägt, unter Bildung des Plasmids pTG2886 LEU2- d, URA3-d. Das Fragment HindIII von 0,9 kb des Plasmids pTG2800, das in der europäischen Patentpublikation EP-A- 0258501 beschrieben ist und das Gen URA3-d trägt, wird dann in die Stelle HindIII dieses Plasmids inseriert unter Bildung von pTG2886 URA3-d, delta LEU2-d. Das Fragment SmaI-BglII von M13TG131 (Kieny et al. (1983), "Gene" 26, 91-99), das mehrere Restriktionsstellen aufweist, wird anschließend in dieses Plasmid eingeführt unter Bildung von pTG3828 (Fig. 4), das umfaßt:Plasmid pTG848 described in European patent publication EP-A-0252854 is digested with BglII, then religated to form pTG2886. The large HindIII-EcoRI fragment of pTG2886 is ligated in the presence of ligase T4 with the 2.1 kb HindIII-EcoRI fragment of plasmid pFL1 (S.A. Parent et al. (1985), "Yeast", 1, 83-138) carrying the sequence of plasmid 2 u of S. cerevisiae to form plasmid pTG2886 LEU2-d, URA3-d. The 0.9 kb HindIII fragment of the plasmid pTG2800, described in European patent publication EP-A-0258501 and carrying the URA3-d gene, is then inserted into the HindIII site of this plasmid to form pTG2886 URA3-d, delta LEU2-d. The SmaI-BglII fragment of M13TG131 (Kieny et al. (1983), "Gene" 26, 91-99), which has several restriction sites, is then introduced into this plasmid to form pTG3828 (Fig. 4), which comprises:
- die Sequenz des Gens URA3, die aus seinem Promotor deletiert worden ist (URA3-d),- the sequence of the URA3 gene deleted from its promoter (URA3-d),
- Restriktionsstellen, die aus M13TG131 stammen, welche die Insertion von Elementen zur Expression eines heterologen Gens, im vorliegenden Fall rHV2Lys47, erlauben,- restriction sites originating from M13TG131, which allow the insertion of elements for the expression of a heterologous gene, in this case rHV2Lys47,
- der Transkritpions-Terminator des Hefe-Gens PGK,- the transcription terminator of the yeast gene PGK,
- ein Fragment von pBR322, das die Replikation und Selektion bei E. coli erlaubt,- a fragment of pBR322 that allows replication and selection in E. coli,
- ein Fragment des Plasmid 2 u, das die für die Replikation und die mitotische Teilung in gleiche Teile in der Hefe erforderlichen Strukturelemente aufweist.- a fragment of plasmid 2u containing the structural elements required for replication and mitotic division into equal parts in yeast.
Das Fragment SphI-SalI des Vektors M13TG3845 (Fig. 3) wird in das Plasmid pTG3828 eingeführt, mit Sphl und Sall digeriert unter Bildung des Expressionsvektors pTG3864 (Fig. 5).The SphI-SalI fragment of the vector M13TG3845 (Fig. 3) is introduced into the plasmid pTG3828, digested with SphI and SalI to form the expression vector pTG3864 (Fig. 5).
Um die für den Vorläufer des Gens MFα1 codierende Sequenz vollständig zu unterdrücken, führt man eine gerichtete Mutagenese bei M13TG3845 (Fig. 3) mit Hilfe des folgenden Oligonucleotids durch:To completely suppress the sequence coding for the precursor of the MFα1 gene, a site-directed mutagenesis is performed on M13TG3845 (Fig. 3) using the following oligonucleotide:
5' GCCTCCCAAGTTTCAGCTATTACGTATACAGACTGC5' GCCTCCCAAGTTTCAGCTATTACGTATACAGACTGC
wobei man den Vektor M13TG3846 erhält. Das Fragment SphI- SalI dieses Vektors wird in das Plasmid pTG3828 (Fig. 4) eingeführt, digeriert mit Sphl und Sall unter Bildung des Expressionsvektors pTG3867. In diesem Vektor sind die Sequenz XI und diejenige, die für rHV2Lys47 codiert, benachbart zueinander angeordnet. Um die schematische Struktur dieses Plasmids zu erhalten, genügt es in der Fig. 5, die Sequenz "pro" zu entfernen, die aus MFα1 deletiert worden ist.obtaining the vector M13TG3846. The SphI-SalI fragment of this vector is introduced into the plasmid pTG3828 (Fig. 4) and digested with SphI and SalI to form the expression vector pTG3867. In this vector, the XI sequence and that coding for rHV2Lys47 are arranged adjacent to each other. To obtain the schematic structure of this plasmid, it is sufficient to remove the "pro" sequence deleted from MFα1 in Fig. 5.
In der Sequenz, die für das Fragment "pro" des Vorläuf ers des Faktors α codiert, wird eine Stelle 5mal geschaffen durch gerichtete Mutagenese an M13TG3841 (Fig. 2) mit Hilfe des folgenden Oligonucleotids:In the sequence encoding the "pro" fragment of the precursor of factor α, a site is created 5 times by site-directed mutagenesis on M13TG3841 (Fig. 2) using the following oligonucleotide:
5' TCCGCATTAGCTGCTCCCGGGAACACTACAACAGAA5' TCCGCATTAGCTGCTCCCGGGAACACTACAACAGAA
wobei man M13TG3869 erhält. Dieser Vektor wird anschließend durch Sphl und SmaI digeriert, das kleine Fragment wird isoliert und es wird mit dem großen Fragment SphI und SmaI von M13TG3845 (Fig. 3) ligiert unter Bildung des Vektors M13TG3891. Das Fragment SphI-SalI dieses Vektors wird in das Plasmid pTG3828 (Fig. 4) eingeführt, das an den Stellen SphI und SalI offen ist, unter Bildung des Expressionsvektors pTG3894, der somit die mutierte Sequenz "pro" des Vorläufers des Faktors α enthält. Um die schematische Struktur dieses Plasmids zu erhalten, genügt es in der Fig. 5, die Sequenz "pro", die aus MFα1 deletiert worden ist, durch die mutierte Sequenz "pro" zu ersetzen.to obtain M13TG3869. This vector is then digested with Sphl and SmaI, the small fragment is isolated and it is ligated with the large SphI and SmaI fragment of M13TG3845 (Fig. 3) to form the vector M13TG3891. The SphI-SalI fragment of this vector is introduced into the plasmid pTG3828 (Fig. 4) open at the SphI and SalI sites to form the expression vector pTG3894, which thus contains the mutated "pro" sequence of the precursor of factor α. To obtain the schematic structure of this plasmid, it is sufficient to replace the "pro" sequence deleted from MFα1 with the mutated "pro" sequence in Fig. 5.
Die Sequenz XI wird durch gerichtete Mutagenese mit M13TG3845 unter Verwendung des folgenden Oligonucleotids modifiziert:Sequence XI is modified by site-directed mutagenesis with M13TG3845 using the following oligonucleotide:
5' GTTTCTCTACTACACTCGCTACTGC5' GTTTCTCTACTACACTCGCTACTGC
Die Modifikation einer einzigen Base ergibt die Sequenz XII und induziert den Ersatz eines Valins durch ein Leucin als Aminosäure R&sub4; des Signal-Peptids. Man erhält so den Bacteriophagen MI3TG3846. Das Fragment SphI-SalI von M13TG3846 wird in das Plasmid pTG3828 (Fig. 4) eingeführt, das an den Stellen SphI und SalI offen ist, unter Bildung des Expressionsvektors pTG3884, der umfaßt:Modification of a single base gives the sequence XII and induces the replacement of a valine by a leucine as amino acid R4 of the signal peptide. The bacteriophage MI3TG3846 is thus obtained. The SphI-SalI fragment of M13TG3846 is introduced into the plasmid pTG3828 (Fig. 4), open at the SphI and SalI sites, to form the expression vector pTG3884, which comprises:
- die Sequenz des Gens URA3, die aus seinem Promotor deletiert worden ist (URA3-d),- the sequence of the URA3 gene deleted from its promoter (URA3-d),
- den Promotor des Gens MFα1, gefolgt von einem Codon ATG als Fragment (a),- the promoter of the gene MFα1, followed by a codon ATG as a fragment (a),
- die Sequenz XII als Fragment (b),- sequence XII as fragment (b),
- die Sequenz "pro", die aus dem Gen MFα1 deletiert worden ist, gefolgt von Codons, die für Lys-Arg codierren, als Fragment (b'),- the sequence "pro" deleted from the gene MFα1, followed by codons coding for Lys-Arg, as fragment (b'),
- die Sequenz, die für rHV2Lys47 codiert, als Fragment (c),- the sequence encoding rHV2Lys47 as a fragment (c),
- den Terminator des Gens, das für PGK der Hefe Codiert,- the terminator of the gene encoding yeast PGK,
- ein Fragment von pBR322,- a fragment of pBR322,
- ein Fragment des Plasmids 2 u.- a fragment of plasmid 2 u.
Ein Hefestamm der Species Saccharomyces cerevisiae vom Genotyp MATα, ura3-251,-373,-328, leu2-3,-112, his3, pep4-3 wird durch die Plasmide pTG3864, pTG3867, pTG3894 und pTG3884 transformiert nach dem Lithiumacetat-Verfahren (H. Ito et al., "J. Bacteriol." (1983), 153: 163) und die Prototrophen Ura+ werden selektioniert. Sie werden anschließend in einem Erlenmeyer-Kolben bei 30ºC auf einem seiektiven Medium (0,7 % Stickstoff-haltige Basen für Hefen (Yeast Nitrogen Base), 0,5 % Casaminosäuren und 1 % Glucose) kultiviert. Nach 48-stündiger Kultivierung trennt man die Zellen und den Überstand durch Zentrifugieren und bestimmt die Thrombin-Inhibitor-Aktivität in dem Überstand unter Anwendung des colorimetrischen Tests (proteolytische Aktivität auf einem synthetischen Substrat, dem Chromozyin TH - Boehringer Mannheim). In der folgenden Tabelle I sind die Analysenergebnisse angegeben; jeder Wert entspricht dem Mittelwert von zwei voneinander unabhängigen Versuchen. Die Aktivität von rHV2Lys47 ist in ATU/ml Überstand ausgedrückt. Tabelle I A yeast strain of the species Saccharomyces cerevisiae of the genotype MATα, ura3-251,-373,-328, leu2-3,-112, his3, pep4-3 is transformed by the plasmids pTG3864, pTG3867, pTG3894 and pTG3884 according to the lithium acetate method (H. Ito et al., "J. Bacteriol." (1983), 153: 163) and the prototrophs Ura+ are selected. They are then cultured in an Erlenmeyer flask at 30ºC on a selective medium (0.7% yeast nitrogen base, 0.5% casamino acids and 1% glucose). After 48 hours of cultivation, the cells and the supernatant are separated by centrifugation and the thrombin inhibitory activity in the supernatant is determined using the colorimetric test (proteolytic activity on a synthetic substrate, Chromozyin TH - Boehringer Mannheim). The results of the analysis are given in Table I below; each value corresponds to the mean of two independent experiments. The activity of rHV2Lys47 is expressed in ATU/ml of supernatant. Table I
In allen Fällen mißt man eine Antithrombin-Aktivität. Das durch die Hefe gebildete Protein rHV2Lys47 wird dann in den Überstand exkretiert. Darüber hinaus wird es in aktiver Form sekretiert. Die besten Ergebnisse erhält man mit den durch pTG3884 und pTG3867 transformierten Stämmen.In all cases, antithrombin activity is measured. The protein rHV2Lys47 produced by the yeast is then excreted into the supernatant. In addition, it is secreted in active form. The best results are obtained with the strains transformed by pTG3884 and pTG3867.
Der Gehalt der Überstände an Protein wird durch HPLC analysiert. Der dabei erhaltene Hauptpeak entspricht demjenigen von rhv2Lys47 (in seiner Form mit 65 Aminosäuren) und die Bestimmung der N-terminalen Sequenz bestätigt, daß ein korrekt synthetisiertes Molekül erhalten wurde.The protein content of the supernatants is analyzed by HPLC. The main peak obtained corresponds to that of rhv2Lys47 (in its 65 amino acid form) and the determination of the N-terminal sequence confirms that a correctly synthesized molecule has been obtained.
Die Synthese wird in zwei mit ihren kohäsiven Enden Kpnl miteinander verbundenen Blöcken durchgeführt. Der erste Block umfaßt 3 Oligonucleotide mit den Nummern 1 bis 3 und der zweite Block umfaßt 6 Oligonucleotide mit den Nummern 4 bis 9. Ihre Sequenz und die Position der Oligonucleotide (letzte Zeile der Tabelle, die Kreise repräsentieren den 5-Abschnitt des Oligonucleotids) sind in der Tabelle II angegeben. Tabelle II Nr. SequenzThe synthesis is carried out in two blocks linked by their cohesive ends Kpnl. The first block comprises 3 oligonucleotides numbered 1 to 3 and the second block comprises 6 oligonucleotides numbered 4 to 9. Their sequence and the position of the oligonucleotides (last row of the table, the circles represent the 5-section of the oligonucleotide) are given in Table II. Table II No. Sequence
Die erhaltene Sequenz ist die folgende: The resulting sequence is the following:
Bei der Synthese des ersten Blockes werden die Oligonucleotide 4, 5, 6, 7, 8 und 9 verwendet und die Synthese wird wie folgt durchgeführt:In the synthesis of the first block, oligonucleotides 4, 5, 6, 7, 8 and 9 are used and the synthesis is carried out as follows:
- die Oligonucleotide 5, 6, 7 und 8 werden zunächst an ihren 5'-Enden phosphoryliert, um die Bildung von Polymeren im Verlaufe des Zusammenbaus zu vermeiden. Für jedes dieser Oligonucleotide werden 100 pmol mit der Polynucleotid-Kinase behandelt, zwei Einheiten in einem Endvolumen von 20 ul von 60 um Tris HCl bei pH 7,5; 10 uM MgCl&sub2;; 8 uM Dithiothreit (Kinations-Puffer), enthaltend 3,3 pmol ATPγ, das mit ³²P (5000 Ci/mmol) markiert ist. Nach 15-minütiger Inkubation bei 37ºC werden 5 umol nichtmarkiertes ATP hinzugefügt;- oligonucleotides 5, 6, 7 and 8 are first phosphorylated at their 5' ends to avoid the formation of polymers during assembly. For each of these oligonucleotides, 100 pmol of polynucleotide kinase are treated, two units in a final volume of 20 µl of 60 µM Tris HCl at pH 7.5; 10 µM MgCl₂; 8 µM dithiothreitol (kination buffer) containing 3.3 pmol of ATPγ labeled with 32P (5000 Ci/mmol). After incubation for 15 minutes at 37°C, 5 µmol of unlabeled ATP are added;
- nach 30-minütiger Inkubation bei 37ºC werden 75 pmol der Oligonucleotide 5, 6, 7 und 8 miteinander gemischt, 3 min lang auf 95ºC erwärmt, dann werden die Oligonucleotide 4 und 9 in einem Endvolumen von 90 umol des vorstehend beschriebenen Kinations-Puffers hinzugegeben. Das Ganze wird 3 min lang auf 95ºC erwärmt, dann innerhalb von 2 h langsam auf 37ºC abgekühlt;- after incubation for 30 minutes at 37ºC, 75 pmol of oligonucleotides 5, 6, 7 and 8 are mixed together, heated to 95ºC for 3 minutes, then oligonucleotides 4 and 9 are added in a final volume of 90 pmol of the kination buffer described above. The whole is heated to 95ºC for 3 minutes, then slowly cooled to 37ºC over 2 hours;
25 pmol dieser hybridisierten Oligonucleotide werden 1 h lang bei 15ºC mit Ligase T4 behandelt. Diese Reaktionsmischung (l pmol) wird anschließend zu 50 ng des Bacteriophagen MI3TGI3I (M.P. Kieny et al. (1983), "Gene", 26 91-99), hinzugegeben und mit EcoRI und Kpnl (1 h bei 15ºC) behandelt. Die Ligationsmischung wird verwendet zur Transformation der kompetenten Zellen des Stammes E. coli JM103 (J. Messing et al. (1981), "Nucleic Acid Res.", 2, 309). Ein die gesuchte Sequenz aufweisender Klon wird isoliert und als M13TG3821 bezeichnet.25 pmol of these hybridized oligonucleotides are treated with ligase T4 for 1 h at 15°C. This reaction mixture (1 pmol) is then added to 50 ng of the bacteriophage MI3TGI3I (M.P. Kieny et al. (1983), "Gene", 26 91-99) and treated with EcoRI and KpnI (1 h at 15°C). The ligation mixture is used to transform competent cells of the E. coli JM103 strain (J. Messing et al. (1981), "Nucleic Acid Res.", 2, 309). A clone containing the sequence of interest is isolated and designated M13TG3821.
Bei der Synthese des zweiten Blockes werden die Oligonucleotide 1, 2 und 3 verwendet und die Synthese wird nach dem gleichen Verfahren wie für die Synthese des ersten Blockes beschrieben durchgeführt. In diesem Fall wird nur das Oligonucleotid 2 an seinem 5'-Ende phosphoryliert.In the synthesis of the second block, oligonucleotides 1, 2 and 3 are used and the synthesis is carried out following the same procedure as described for the synthesis of the first block. In this case, only oligonucleotide 2 is phosphorylated at its 5'-end.
Dieser zweite Block wird zwischen die Stellen HindIII und KpnI des Bacteriophagen M13TG3821 kloniert und es wird ein Klon isoliert, der die DNA-Sequenz trägt, die für das Defensin A codiert (Fig. 5), er wird als MI3TG3849 bezeichnet.This second block is cloned between the HindIII and KpnI sites of the bacteriophage M13TG3821 and a clone carrying the DNA sequence encoding defensin A (Fig. 5) was isolated; it is designated MI3TG3849.
Das Fragment SphI - SmaI von 1045 Basenpaaren des Bacteriophagen M13TG3846, wie es vorstehend beschrieben wurde (Beispiel 1, E), wird in den vorstehend beschriebenen Vektor MI3TG3869 (Beispiel 1, D) transferiert, der vorher mit SphI und SmaI digeriert worden ist. Man erhält so den Vektor M13TG4803, der trägt:The 1045 base pair SphI - SmaI fragment of the bacteriophage M13TG3846 described above (Example 1, E) is transferred into the vector MI3TG3869 described above (Example 1, D) which has been previously digested with SphI and SmaI. This gives the vector M13TG4803 which carries:
- den Promotor des Gens MFal, gefolgt von einem Codon ATG,- the promoter of the gene MFal, followed by a codon ATG,
- die Sequenz XII,- the sequence XII,
- die mutierte Sequenz "pro" des Gens MFal, gefolgt von Codons, die für Lys-Arg codieren,- the mutated sequence "pro" of the MFal gene, followed by codons coding for Lys-Arg,
- die Sequenz, die für rHV2Lys47 codiert.- the sequence encoding rHV2Lys47.
Um die für rHV2Lys47 codierende Sequenz durch diejenige zu ersetzen, die für das Insekten-Defensin A codiert, führt man in die mutierte Sequenz von MFα1, die für "pro" codiert, mit Hilfe des folgenden Oligonucleotids eine Stelle HindIII ein:To replace the sequence coding for rHV2Lys47 with that coding for insect defensin A, a HindIII site is introduced into the mutated sequence of MFα1 coding for "pro" using the following oligonucleotide:
5' GAAGGGGTAAGCTTGGATAAA5' GAAGGGTAAGCTTGGATAAA
Dann führt man das oben beschriebene Fragment HindIII - BamHI von M13TG3849 (Beispiel 3, A), das die für das Defensin A codierende synthetische Sequenz trägt, in diesen Vektor ein, der vorher mit HindIII und BamHI behandelt worden ist, um die für rHV2Lys47 codierende Sequenz zu eliminieren.Then, the HindIII-BamHI fragment of M13TG3849 described above (Example 3, A), which carries the synthetic sequence coding for defensin A, is introduced into this vector, which has previously been treated with HindIII and BamHI to eliminate the sequence coding for rHV2Lys47.
Das Fragment SphI-SalI von M13TG4813 wird in das Plasmid pTG3828 (Fig. 4) eingeführt, das an den Stellen SphI und SalI offen ist, unter Bildung des Expressionsvektors pTG4839, der umfaßt:The SphI-SalI fragment of M13TG4813 is introduced into the plasmid pTG3828 (Fig. 4) open at the SphI and SalI sites, to form the expression vector pTG4839, which comprises:
- die Sequenz des Gens URA3, die aus ihrem Promotor deletiert worden ist (URA3-d),- the sequence of the URA3 gene deleted from its promoter (URA3-d),
- den Promotor des Gens MFα1, gefolgt von einem ATG- the promoter of the gene MFα1, followed by an ATG
- die Sequenz XII,- the sequence XII,
- die mutierte Sequenz "pro" des Gens MFα1, gefolgt von Codons, die für Lys-Arg codieren,- the mutated sequence "pro" of the MFα1 gene, followed by codons coding for Lys-Arg,
- die synthetische Sequenz, die für das Insekten-Defensin A codiert,- the synthetic sequence encoding insect defensin A,
- den Terminator des Gens, das für PGK von Hefe codiert,- the terminator of the gene encoding yeast PGK,
- ein Fragment von pBR322,- a fragment of pBR322,
- ein Fragment des Plasmids 2u.- a fragment of plasmid 2u.
Ein Hefestamm der Species Saccharomyces cerevisiae vom Genotyp MATα, ura3-251,-373,-328, leu2-3,-112, his3, pep4-3 wird durch das Plasmid pTG4839 nach dem Lithiumacetat-Verfahren (H. Ito et al., "J. Bacteriol." (1983), 153: 163) transformiert und die Prototrophen Ura+ werden selektioniert. Anschließend werden sie in einem Erlenmeyer-Kolben bei 30ºC auf einem selektiven Medium (0,7 % stickstoffhaltige Basen für Hefen (Yeast Nitrogen Base), 0,5 % Casaminosäuren und 1 % Glucose) kultiviert. Nach 48-stündiger Kultivierung trennt man die Zellen und den Überstand durch Zentrifugieren und der Überstand wird durch ein 22 u-Filter filtriert, dann durch eine Sep-Pak C18-Kartusche laufen gelassen. Das fixierte Material wird mit 60 % Acetonitril, 0,1 % Trifluoroessigsäure in Wasser eluiert und unter Vakuum getrocknet. Die antibakterielle Aktivität des Defensins A wird anschließend unter Verwendung eines Ausstreich-Tests auf Agar oder auf Agar-Agar, in das Bakterienkeime eingeimpft worden sind (Micrococcus luteus) nach dem von Lambert et al. (1989) in "PNAS" 86: 262-266, beschriebenen Verfahren nachgewiesen.A yeast strain of the species Saccharomyces cerevisiae of the genotype MATα, ura3-251,-373,-328, leu2-3,-112, his3, pep4-3 is transformed by the plasmid pTG4839 using the lithium acetate method (H. Ito et al., "J. Bacteriol." (1983), 153: 163) and the prototrophs Ura+ are selected. They are then cultured in an Erlenmeyer flask at 30ºC on a selective medium (0.7% yeast nitrogen base, 0.5% casamino acids and 1% glucose). After 48 hours of cultivation, the cells and supernatant are separated by centrifugation and the supernatant is filtered through a 22 u filter, then passed through a Sep-Pak C18 cartridge. The fixed material is eluted with 60% acetonitrile, 0.1% trifluoroacetic acid in water and dried under vacuum. The antibacterial activity of defensin A is then demonstrated using a streak test on agar or on agar-agar inoculated with bacteria (Micrococcus luteus) according to the procedure described by Lambert et al. (1989) in "PNAS" 86: 262-266.
In den Überständen der durch das Plasmid pTG4839 transformierten Hefen wird effektiv eine antibakterielle Aktivität nachgewiesen. Das durch die Hefe gebildete Protein Defensin A wird somit in den Überstand exkretiert. Darüber hinaus wird es in der aktiven Form sekretiert.Antibacterial activity is effectively detected in the supernatants of yeasts transformed by the plasmid pTG4839. The protein defensin A produced by the yeast is thus excreted into the supernatant. In addition, it is secreted in the active form.
Der proteingehalt der Überstände wird durch HPLC analysiert. Der erhaltene Hauptpeak entspricht demjenigen des Insekten-Defensins A und die Bestimmung der Protein-Sequenz bestätigt, daß ein korrekt synthetisiertes Molekül erhalten worden ist.The protein content of the supernatants is analyzed by HPLC. The main peak obtained corresponds to that of insect defensin A and the determination of the protein sequence confirms that a correctly synthesized molecule has been obtained.
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PCT/FR1990/000306 WO1990013646A1 (en) | 1989-04-28 | 1990-04-27 | Application of novel dna fragments as a coding sequence for a signal peptide for the secretion of mature proteins by recombinant yeast, expression cassettes, transformed yeasts and corresponding process for the preparation of proteins |
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FR2669931B1 (en) * | 1990-11-29 | 1995-03-31 | Transgene | NEW VARIANTS DERIVED FROM INTERFERONS-ALPHA AND THEIR PRODUCTION PROCESS. |
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FR2700338B1 (en) * | 1993-01-11 | 1995-03-31 | Transgene Sa | New pro sequence allowing the secretion of heterologous proteins from a yeast cell. |
WO1995001431A1 (en) * | 1993-06-29 | 1995-01-12 | Transgene S.A. | Peptide family, designated as xenonins |
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US5712114A (en) * | 1995-06-06 | 1998-01-27 | Basf Aktiengesellschaft | Compositions for expression of proteins in host cells using a preprocollagen signal sequence |
US5837247A (en) * | 1995-06-16 | 1998-11-17 | United States Of America As Represented By The Public Health Service National Institutes Of Health | Chemotactic agents for t-cells |
US6998116B1 (en) | 1996-01-09 | 2006-02-14 | Genentech, Inc. | Apo-2 ligand |
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US6469144B1 (en) | 1996-04-01 | 2002-10-22 | Genentech, Inc. | Apo-2LI and Apo-3 polypeptides |
US5851984A (en) * | 1996-08-16 | 1998-12-22 | Genentech, Inc. | Method of enhancing proliferation or differentiation of hematopoietic stem cells using Wnt polypeptides |
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ATE78294T1 (en) * | 1984-03-27 | 1992-08-15 | Transgene Sa | EXPRESSION VECTORS FOR HIRUDIN, TRANSFORMED CELLS AND METHODS OF PRODUCTION OF HIRUDIN. |
FR2593518B1 (en) * | 1985-05-02 | 1989-09-08 | Transgene Sa | VECTORS FOR THE EXPRESSION AND SECRETION OF HIRUDIN BY TRANSFORMED YEASTS |
MY101203A (en) * | 1985-12-12 | 1991-08-17 | Ucp Gen Pharma Ag | Production of thrombin iinhibitors. |
MC1834A1 (en) * | 1986-07-10 | 1988-06-03 | Transgene Sa | DNA FUNCTIONAL BLOCK AND PLASMID ENCODING HIRUDINE, TRANSFORMED YEAST, PROCESS FOR PREPARING HIRUDINE, HIRUDINE OBTAINED AND ITS USE |
FR2607517B2 (en) * | 1986-12-01 | 1989-12-22 | Transgene Sa | VECTORS FOR EXPRESSING HIRUDIN VARIANTS IN YEAST, PROCESS AND PRODUCT OBTAINED |
FR2628429B1 (en) * | 1988-03-08 | 1990-12-28 | Transgene Sa | HIRUDINE VARIANTS, USES THEREOF AND PROCESSES FOR OBTAINING THEM |
FR2633296B1 (en) * | 1988-06-24 | 1991-11-15 | Centre Nat Rech Scient | PEPTIDE MOLECULES WITH ACTION ON GRAMS WITH A POSITIVE GRAM |
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